CN111484544A

CN111484544A - Polypeptide for treating sepsis and application thereof

Info

Publication number: CN111484544A
Application number: CN201910082502.6A
Authority: CN
Inventors: 赵树民; 石松传; 鲍勇刚; 威廉姆·坎贝尔
Original assignee: Beijing Pepnoch Biotech Corp ltd
Current assignee: Beijing Pepnoch Biotech Corp ltd
Priority date: 2019-01-28
Filing date: 2019-01-28
Publication date: 2020-08-04
Anticipated expiration: 2039-01-28
Also published as: CN111484544B

Abstract

The invention relates to a polypeptide PEP-C1 for treating sepsis, wherein the amino acid sequence of the polypeptide PEP-C1 is Asp-Ser-L eu-Trp-Asn-Ile-Pro.

Description

Polypeptide for treating sepsis and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a polypeptide for treating sepsis and application thereof.

Background

Sepsis refers to the systemic inflammatory response syndrome clinically caused by pathogenic infection. Although sepsis is caused by infection, once it occurs, its development follows its own pathological course and laws, so it is essentially the body's response to infectious agents. Clinically, bacteria commonly caused by surgical trauma, low immunity and the like enter blood circulation, grow and reproduce in the blood circulation and generate toxin to cause severe systemic infection, and further can be developed into septic shock and multi-organ failure. Sepsis has a mortality rate of 30% -70%, over 1800 million severe sepsis cases worldwide per year, and this figure also rises at a rate of 1.5% -8.0% per year. The treatment cost of the sepsis is high, the medical resource consumption is large, the life quality of human beings is seriously influenced, great threat to the health of the human beings is already caused, and no effective medicine is used for treating the sepsis so far, so that the prevention and treatment medicine for treating the sepsis is urgently needed in clinic.

Sepsis involves a number of factors including complex systemic inflammatory network effects, immune dysfunction, abnormal blood clotting functions, and abnormal host responses to various pathogens. Recent studies have found that the complement system plays an important role in the pathogenesis of sepsis. Excessive activation of complement system exists in the initial stage of sepsis, which results in the rise of the level of plasma C5a and induces the abnormal expression of the receptor C5aR on cells, and the excessive action between C5a and C5aR causes damage to the organism and causes severe disease manifestations such as septic shock and the like. C5a and C5aR play an important role in the regulation of inflammatory mediators, so that the screening or design of a molecular inhibitor or an antibody medicament for treating sepsis by using C5a and C5aR as targeting proteins is very valuable and promising.

Polypeptide drugs are a hotspot for research and development of new drugs in the world in recent years, and are also one of the key directions for biological medicine research in China. Compared with the traditional medicine, the polypeptide medicine has the following obvious advantages: (1) the activity is high, and the remarkable high activity can be shown at very low dosage and concentration; (2) the molecular weight is small, and compared with protein, the protein is easy to artificially and chemically synthesize, and the structure is convenient to modify; (3) the synthesis efficiency is high. The technological progress in recent years makes the solid phase synthesis of polypeptide become simple, the process is automatic, easy to control; (4) has less side effect. Because many polypeptide drugs adopt sequences homologous with human, and the polypeptide drugs have small molecular weight and no antigenicity, and are not easy to cause immune response. The development of a polypeptide medicament for treating sepsis, which has high specificity and good tolerance, is a problem to be solved urgently in clinic.

Disclosure of Invention

Aiming at the condition that the prior art lacks special treatment medicines for sepsis, the invention provides a polypeptide PEP-C1 for treating sepsis aiming at a specific protein target.

According to one aspect of the invention, the invention provides a polypeptide PEP-C1 for treating sepsis, which has an amino acid sequence of Asp-Ser-L eu-Trp-Asn-Ile-Pro (SEQ ID NO: 1).

According to another aspect of the invention there is provided the use of the polypeptide in the manufacture of a medicament for the treatment of sepsis.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the active ingredient polypeptide PEP-C1 and a pharmaceutically acceptable carrier.

The pharmaceutical composition includes, but is not limited to, injection, tablet, granule, capsule, oral liquid, pill, etc.

The pharmaceutically acceptable carrier includes diluent, excipient, filler, binder, wetting agent, disintegrating agent, absorption enhancer, surfactant, adsorption carrier, lubricant, etc. which are conventional in the pharmaceutical field, and flavoring agent, sweetener, etc. may be added if necessary.

The polypeptide of the present invention should be present in an "effective amount" as an active ingredient, which refers to a non-toxic, but sufficient amount of a drug or agent that provides the desired effect. In the pharmaceutical compositions of the present invention, an "effective amount" of an ingredient refers to an amount of that ingredient which, when used in combination with other ingredients, is effective in providing the desired effect. The "effective amount" will vary from subject to subject, depending on age and general condition of the individual, the particular active agent, and the like. Thus, an exact "effective amount" cannot always be intended, however, a suitable "effective amount" in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

The polypeptide of the present invention can be prepared by methods known to those skilled in the art (e.g., solid phase synthesis), and can be isolated and purified by separation and purification methods known to those skilled in the art (e.g., high performance liquid chromatography).

The invention has the beneficial effects that C5a and C5aR molecules play a very key role in a signal path related to sepsis, the invention is based on C5a target protein, a proprietary polypeptide molecular system is applied, a C5a protein molecule three-dimensional structure is simulated, molecular docking, homologous modeling, combinatorial library design, chemical informatics, de novo design, structural biology, biomacromolecule simulation, pharmacophore, ADME analysis and other molecular and drug design methods are applied, C5a protein is used as a target molecule, and a plurality of specific polypeptides capable of blocking C5a-C5aR combination are designed, a polypeptide PEP-1 with remarkable treatment effect on sepsis is screened out through a series of activity verification tests, and the research result shows that the polypeptide PEP-C1 shows good effect in the process of treating a cecal intestine ligation puncture model (C L P) sepsis mouse, and has good stability.

Drawings

FIG. 1 is an MS map of the polypeptide PEP-C1;

FIG. 2 is an HP L C map of the polypeptide PEP-C1;

FIG. 3 is a graph of the effect of a polypeptide on the sepsis mouse cytokine TNF α;

FIG. 4 shows the effect of polypeptides on sepsis mouse cytokine I L-1 β;

FIG. 5 shows the effect of the polypeptide on the cytokine MIP-2 of septic mice.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Furthermore, it should be understood that various changes or modifications can be made by those skilled in the art after reading the description of the present invention, and such equivalents also fall within the scope of the invention.

EXAMPLE 1 chemical Synthesis of the polypeptide PEP-C1

Synthesis Polypeptides were synthesized from C-terminus to N-terminus using solid phase synthesis. The synthesis is carried out by adopting a chemical synthesizer (AMS586Multiple Peptide synthesizer, ABIMED, Germany), amino acid protected by Fmoc is used as raw material, Fmoc-Rink linker resin is used as adhesion matrix, HOBT is used as condensing agent, DIC is used as activating agent, and polypeptide is synthesized layer by layer.

During the synthesis, 2% acetic anhydride solution in DMF is used as side chain blocking reagent, 20% piperidine is used as Fmoc removing reagent, TFA cutting and side chain radical removing operation are carried out after the synthesis, the synthesized crude product is collected by centrifugation, and R-HP L C (Waters 741) and C are used₁₈-column(Waters Delta-pak^TM40 x 100mm, 15um, 100 angstroms) to obtain polypeptide with purity of more than 98%, and freeze-drying into powder.

The MS pattern and the HP L C pattern of the polypeptide Asp-Ser-L eu-Trp-Asn-Ile-Pro are respectively shown in figures 1 and 2, as shown in the figure, the polypeptide has a characteristic peak at 7.698min, and the molecular weight is 843.1 (the theoretical molecular weight is 843.93).

The HP L C chromatographic conditions were:

mobile phase: mobile phase A: an aqueous solution containing 0.1% trifluoroacetic acid; mobile phase B: acetonitrile (80%) with 0.1% trifluoroacetic acid-water (20%);

elution procedure: 0 → 20min, mobile phase B38% → 58%;

flow rate: 1 ml/min;

detection wavelength: 220 nm;

retention time: and 20 min.

Example 2 Activity assay of the polypeptide PEP-C1

The animal experiment research of the sepsis at present is mainly based on the animal model of sepsis of inducing the method, wherein the cecal ligature perforation model of the mouse (C L P), because the animal is cheap and easy to operate, advantage such as being simple is applied widely, this research will utilize C L P to set up the model of mouse sepsis, through evaluating the change of indexes such as survival rate of mouse, determination of cytokine level (TNFa, I L-1 β, MIP-2), etc., the pathogenesis of sepsis induced by C L P is studied, offer the technical support for carrying on the anti-inflammatory drug screening in the later stage, this research uses this model to carry on the research of polypeptide PEP-C1 to sepsis.

1 method of experiment

1.1 cecal ligation puncture model (C L P) establishment

C57B L/6 mouse is anesthetized, lies on the back and is fixed on an operating table, the abdomen is disinfected by 75% alcohol, the lower finger of the xiphoid process is opened with a scalpel along the midline of the abdomen for about 2cm, the cecum is carefully separated, the position 1/2 of the cecum is tied by a 3-line, after tying, an 8-line needle is used for puncturing the intestinal wall for 2 times at the position about 0.5cm above the cecum of the cecum, a proper amount of intestinal content is extruded out, the intestinal content and the cecum are put back to the abdominal cavity in situ, and suturing layer by layer is carried out.

1.2 group settings

The experiment was divided into 6 groups, namely blank group, sham group, model group, positive drug (imipenem/cilastatin) group, polypeptide I group and polypeptide II group.

1.3 dosage by administration

333.33mg/kg of positive drug (imipenem/cilastatin), 6mg/kg of dosage of polypeptide I, target polypeptide Asp-Ser-L eu-Trp-Asn-Ile-Pro, and 6mg/kg of dosage of polypeptide II, 12 mg/kg. of dosage of target polypeptide Asp-Ser-L eu-Trp-Asn-Ile-Pro, the first administration after 3 hours of operation, intraperitoneal injection, injection of physiological saline with the same amount in a model group and a sham operation group, and administration or physiological saline once every 12 hours after the first administration.

1.4 Observation index

1.4.1C L P postoperative mouse survival rate, when observing survival rate index, the drug dose is unchanged, once every 12h, until the mouse dies or the drug administration is completed for 3 days, and the survival rate of each group of mice is observed every 7d after operation.

1.4.2C L P postoperative mouse cytokine level, taking blood after 36h of decapitation, separating serum from each group of surviving mice, and detecting the contents of TNF α, I L-1 β and MIP-2 by an E L ISA method.

2 results of the experiment

2.1 survival of mice per group 7 days post-surgery

The results are shown in Table 1.

TABLE 1 Effect of Polypeptides on survival of septic mice

The results in table 1 show that the model group sepsis mice had a very high mortality rate (up to 83%); the mortality of sepsis mice after the intervention of the positive drug is obviously controlled; the mortality rate of the sepsis mice is obviously reduced by using the low dose (6mg/kg) and the high dose (12mg/kg) of the polypeptide PEP-C1 to intervene in the model mice, and the effect of the model mice intervened by using the high dose (12mg/kg) of the polypeptide PEP-C1 is better than that of the positive drug.

2.2 post-operative mouse cytokine levels

2.2.1 measurement of TNF α content in mouse serum

The results are shown in Table 2 and FIG. 3.

TABLE 2 Effect of the Polypeptides on the sepsis mouse cytokine TNF α

Group of	TNFαpg/ml
		Blank group	15.344±3.946
Artificial operation group	15.549±5.697
		Model set	175.007±26.469
Positive drug group	82.945±26.778*
		Group I Polypeptides	121.167±26.940*
Group II Polypeptides	92.899±15.076*

Note: the results were calculated for 10 mice per spot per group. P <0.01 compared to model group;

the result of the content determination of TNF α in the serum of the mice shows that the content of TNF α in a blank group and a sham operation group is in a low normal level, the content of TNF α in a model group is obviously increased, the content of TNF α is obviously controlled after the intervention of positive drugs, the intervention of the model mice by using polypeptide PEP-C1 with low dose (6mg/kg) and high dose (12mg/kg) has obviously reduced content of TNF α, and the effect of the model mice interfered by using polypeptide PEP-C1 with high dose (12mg/kg) is equivalent to that of the positive drugs.

2.2.2 measurement of the level of I L-1 β in mouse serum

The results are shown in Table 3 and FIG. 4.

TABLE 3 Effect of the Polypeptides on sepsis mouse cytokine I L-1 β

Group of	IL-1βpg/ml
		Blank group	56.674±5.603
Artificial operation group	59.163±7.531
		Model set	99.136±7.55
Positive drug group	60.468±5.393#
		Group I Polypeptides	72.838±8.854#
Group II Polypeptides	62.338±7.196#

Note: the results were calculated for 10 mice per spot per group. #: p <0.05 compared to model group

The measurement result of the I L-1 β content in the serum of the mouse shows that the content of I L-1 β in a blank group and a sham operation group is in a low normal level, the content of I L-1 β in a model group is obviously increased, the content of I L-1 β is obviously controlled after the intervention of a positive drug, the content of I L-1 β of the model mouse is obviously reduced by using low-dose (6mg/kg) and high-dose (12mg/kg) polypeptide PEP-C1, and the effect of the model mouse intervened by using the high-dose (12mg/kg) polypeptide PEP-C1 is equivalent to that of the positive drug.

2.2.3 measurement of MIP-2 content in mouse serum

The results are shown in Table 4 and FIG. 5.

TABLE 4 Effect of the Polypeptides on the sepsis mouse cytokine MIP-2

Group of	MIP-2pg/ml
		Blank group	8.266±3.217
Artificial operation group	10.517±2.151
		Model set	168.694±20.987
Positive drug group	104.556±26.917*
		Group I Polypeptides	136.203±24.679#
Group II Polypeptides	109.019±19.92*

Note: the results were calculated for 10 mice per spot per group. #: p <0.05 compared to model group; p <0.01 compared to model group.

The content measurement result of MIP-2 in the serum of the mouse shows that the content of MIP-2 in the blank group and the sham operation group is at a low normal level; the content of model MIP-2 is obviously increased; the content of MIP-2 is obviously controlled after the intervention of positive drugs; the polypeptide PEP-C1 with low dose (6mg/kg) and high dose (12mg/kg) is used for intervening model mice, the MIP-2 content of the model mice is obviously reduced, and the effect of the model mice which are intervened by the polypeptide PEP-C1 with high dose (12mg/kg) is equivalent to that of a positive drug.

3 conclusion

The research results show that main cell inflammation factors TNFa, I L-1 β and MIP-2 of a sepsis mouse using the polypeptide PEP-C1 are obviously reduced compared with a model group, the PEP-C1 has a better treatment effect on the sepsis of the mouse, and the mouse has a comparable effect compared with a positive drug (imipenem/cilastatin, 333.33mg/kg) under the intervention of high-dose PEP-C1(12mg/kg), but the dosage of the PEP-C1 is much lower.

According to the analysis on the mortality data, compared with the model group, the positive drug, the PEP-C1 low dose group (6mg/kg) and the PEP-C1 high dose group (12mg/kg) are obviously reduced, and the PEP-C1 high dose group (12mg/kg) has higher mortality reducing effect than the positive drug group. The results show that the C5a/C5aR blocker has good effect on treating sepsis in animal experiments, and provides a new effective candidate medicament for a new medicament for treating sepsis.

Example 3

Adding appropriate adjuvants into PEP-C110 g, and making into injection by lyophilized powder for injection and sterile packaged powder for injection.

Example 4

Adding appropriate adjuvants into the polypeptide PEP-C110 g, and making into tablet (including sustained release tablet, matrix tablet, coated tablet, dispersible tablet, etc.) by tablet process.

Example 5

Adding appropriate adjuvant into the polypeptide PEP-C110 g, and making into capsule by capsule process.

Example 6

Adding appropriate adjuvant into the polypeptide PEP-C110 g, and making into emulsion by emulsion (including microemulsion, nanoemulsion, etc.).

Example 7

Adding appropriate adjuvant into the polypeptide PEP-C110 g, and making into granule by granule process.

Example 8

Adding appropriate adjuvants into the polypeptide PEP-C110 g, and making into sustained-release controlled-release preparation by sustained-release controlled-release preparation process.

Example 9

Adding appropriate adjuvants into PEP-C110 g, and making into oral liquid by oral liquid process.

Example 10

Adding appropriate adjuvants into the polypeptide PEP-C110 g, and making into liposome by liposome process.

Sequence listing

<110> Beijing Bo peptide unknown Biotechnology Limited

<120> polypeptide for treating sepsis and application thereof

<160>1

<170>PatentIn Version 3.3

<210>1

<211>7

<212>PRT

<213> Artificial sequence

<400>1

Asp Ser Leu Trp Asn Ile Pro 7

Claims

1. A polypeptide for the treatment of sepsis, wherein the amino acid sequence of said polypeptide is:

Asp-Ser-Leu-Trp-Asn-Ile-Pro。

2. use of a polypeptide according to claim 1 in the manufacture of a medicament for the treatment of sepsis.

3. A pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition of claim 3, wherein the pharmaceutical composition comprises an injection, a tablet, a granule, a capsule, an oral liquid, or a pill.

5. Use of a pharmaceutical composition according to claim 3 in the manufacture of a medicament for the treatment of sepsis.